The present invention relates generally to chimeric proteins comprising the extracellular and transmembrane portions of CD4 molecules and an src protein tyrosine kinase. Such CD4 chimeric proteins may amplify the signal produced by T lymphocyte stimulation. These CD4 chimeric proteins may be employed to identify drugs that block CD4.sup.+ T lymphocyte activation. The CD4 chimeric proteins are also useful for identifying self antigens that may mediate autoimmune diseases. Also provided are CD8 chimeric proteins having an src tyrosine kinase linked to the extracellular and transmembrane portions of a CD8 molecule. The CD8 chimeric proteins may be used to screen for MHC Class I restricted antigens. Both the CD4 and CD8 chimeric proteins may be employed in gene therapy treatments to enhance in vivo immunological response to specific antigens.
Cytoplasmic protein tyrosine kinases (PTK's) of the src family have important roles in signal transduction processes in multiple cell types (Bolen et al., Adv. Can. Res., 57:103-149 (1991)). Members of this family share several features: they are attached to cellular membranes through a myristylated N-terminus, they have unique N-terminal domains, and they have homologous SH3, SH2, and catalytic domains (FIG. 1A). Similar SH2 and SH3 domains are found in a wide variety of molecules involved in signal transduction (Koch et al., Science, 252:668-674 (1991)). The SH2 domains interact specifically with various proteins containing phosphotyrosine residues, whereas SH3 regions bind guanine nucleotide releasing factors, potentially linking the PTK's to the ras signaling pathway (Feig, Science, 260:767-768 (1993)). Multiple src family molecules are expressed in most cells and ablation of individual genes has resulted in developmental defects of variable severity (Soriano et al., Cell, 64:693-702 (1991); Molina et al., Nature, 357:161-164 (1992); Appleby et al., Cell, 70:751-763 (1992); Stein et al., Cell, 70:741-750 (1992)). Some functions may be carried out by any one of several src family members, whereas others may only be fulfilled by a single one of these molecules.
Early activation events in T lymphocytes require the triggering of a tyrosine phosphorylation pathway that appears to involve one or more of these molecules (Weiss, Cell, 73:209-212 (1993)). A limited number of these kinases, Lck, Fyn, and Yes, are expressed in T cells. Of these, the best-characterized is the lymphocyte-specific tyrosine kinase, P56.sup.lck (Lck), whose unique N-terminal domain interacts with the cytoplasmic tails of the CD4 and CD8 glycoproteins. These are molecules that bind to surface MHC class II and class I molecules, respectively, and participate with the T cell antigen receptor (TCR) in early events of T cell activation (Rudd et al., Proc. Natl. Acad. Sci. USA, 85:5190-5194 (1988); Veillette et al., Cell, 55:301-308 (1988); Shaw et al., Cell, 59:627-636 (1989); Turner et al., Cell, 60:755-765 (1990); Shaw et al., Mol. Cell. Biol., 10:1853-1862 (1990)). The interaction of Lck with CD4 and CD8 is restricted to this member of the src family and is required for effective antigen-specific responses of several different T cell hybridomas (Zamoyska et al., Nature, 342:278-281 (1989); Glaichenhaus et al., Cell, 64:511-520 (1991)).
Lck apparently has multiple functions that are essential in T cell development and activation. Inactivation of the Lck gene in mice results in early arrest of thymocyte maturation, prior to cell surface expression of CD4, CD8, and the T cell receptor, suggesting that Lck has a critical function early in T cell development that is independent of these cell surface molecules (Molina et al., Nature, 357:161-164 (1992)). In the human T cell leukemic line, Jurkat, absence of Lck results in loss of activation in response to anti-TCR antibodies (Straus and Weiss, Cell, 70:585-593 (1992)). The related PTK's present in developing thymocytes and in Jurkat cells appear unable to substitute for Lck. This may be due to a requirement for Lck to associate with cell surface molecules other than CD4 and CD8 that are involved in early development and in TCR-mediated signaling.
Activation of T lymphocytes upon their encounter with MHC-bound peptide antigens is mediated through a complex machinery associated with the T cell antigen receptor (TCR). The clonally-restricted TCR provides specificity for antigen, while associated non-polymorphic polypeptides are involved in the signal transduction process (Irving and Weiss, Cell, 64:891-901 (1991); Romeo et al., Cell, 68:889-897 (1992); Letourneur and Klausner, Science, 255:79-82 (1992); Wegener et al., Cell, 68:83-95 (1992)).
In addition, the MHC-binding co-receptor molecules, CD4 and CD8, are required for initiating signals, both during thymocyte development and in the activation of mature T cells (Fung-Leung et al., Cell, 65:443-449 (1991); Rahemtulla et al., Nature, 353:180-184 (1991); Killeen et al., EMBO J., 12:1547-1553 (1993)). Apparently, signaling requires coordinate recognition of MHC by the co-receptors and by the TCR. This is achieved by binding of CD4 and CD8 to membrane-proximal domains of class II or class I molecules, respectively, while the TCR binds to the peptide-containing surface (Salter et al., Nature, 345:41-46 (1990); Aldrich et al., Nature, 352:718-721 (1991); Ingold et al., Nature, 352:721-723 (1991); Killeen et al., J. Exp. Med., 176:89-97 (1992); Glaichenhaus et al., Cell, 64:511.varies.520 (1991); Konig et al., Nature, 356:796-798 (1992)).
The signaling cascade that follows engagement of the T cell receptor by antigen is dependent on the activity of cytoplasmic tyrosine kinases (Klausner and Samelson, Cell, 64:875-878 (1991)). Ligation of the TCR results in rapid phosphorylation of intracellular proteins on tyrosine residues (June et al., J. Immunol., 144:1591-1599 (1990)). Inhibitors of PTK function block the early signaling events, notably the phosphorylation and activation of phospholipase C.gamma.1 a key enzyme involved in the generation of second messengers that regulate intracellular free calcium concentration and the activity of protein kinase C (Mustelin et al., Science, 247:1584-1587 (1990)).
Several tyrosine kinases have been implicated in the initiation of the T cell signaling pathway. The lymphoid-specific cytoplasmic PTK, Lck, apparently is a key component in this process: its absence prevents TCR-mediated activation of Jurkat cells (Straus and Weiss, Cell, 70:585-593 (1992)), and PLC-.gamma.1 has been co-precipitated with Lck following activation (Weber et al., J. Exp. Med., 176:373-379 (1992)).
A second src family PTK, Fyn-T, has also been implicated in TCR-mediated activation because it is expressed in a T cell-specific manner. Fyn-T is associated with TCR proteins in cell lysates and its level of expression correlates with the magnitude of thymocyte stimulation. A third kinase that appears to have a role in signaling is ZAP-70, a cytoplasmic PTK that is tightly associated with the TCR-.zeta. chain in activated T cells.
Blocking T cell function is desirable in many instances. For example, blocking T cell activation may provide a means of treating and preventing autoimmune diseases, such as systemic lupus erythematosis, rheumatoid arthritis, Sjogren's syndrome, and the like. Interfering with intracellular signal transduction following antigenic stimulation of T cells could provide a means for reducing excessive inflammation and alleviating many clinical illnesses.
Enhancement of immunological response is desirable in other clinical illnesses. For example, malignancy often impairs immune responses. In these patients, it is desirable to enhance the immune response to help fight infections as well as for primary treatment of the underlying malignancy.
No convenient means have been available to identify drugs that block T cell function at the level of protein tyrosine kinase activity. A means to identify such drugs would provide a marked advance in the art of pharmaceutical development. With such a method, skilled artisans could quickly identify promising compounds for clinical use. Quite surprisingly, the present invention fulfills these and other related needs.